The vegetative insecticidal proteins (Vip), secreted by many Bacillus thuringiensis strains during their vegetative growth stage, are genetically distinct from known insecticidal crystal proteins (ICPs) and represent the second-generation insecticidal toxins. Compared with ICPs, the insecticidal mechanisms of Vip toxins are poorly understood. In particular, there has been no report of a definite receptor of Vip toxins to date. In the present study, we identified the scavenger receptor class C like protein (Sf-SR-C) from the Spodoptera frugiperda (Sf9) cells membrane proteins that bind to the biotin labeled Vip3Aa, via the affinity magnetic bead method coupled with HPLC-MS/MS. We then certified Vip3Aa protoxin could interact with Sf-SR-C in vitro and ex vivo. In addition, downregulation of SR-C expression in Sf9 cells and Spodoptera exigua larvae midgut reduced the toxicity of Vip3Aa to them. Coincidently, heterologous expression of Sf-SR-C in transgenic Drosophila midgut significantly enhanced the virulence of Vip3Aa to the Drosophila larvae. Moreover, the complement control protein domain and MAM domain of Sf-SR-C are involved in the interaction with Vip3Aa protoxin. Furthermore, endocytosis of Vip3Aa mediated by Sf-SR-C correlates with its insecticidal activity. Our results confirmed for the first time that Sf-SR-C acts as a receptor for Vip3Aa protoxin and provides an insight into the mode of action of Vip3Aa that will significantly facilitate the study of its insecticidal mechanism and application. Bacillus thuringiensis Vip3A has potential in control of Lepidopteran pest and has been used in transgenic plants. However, studies of the insecticidal mechanisms of Vip3A are rare, and none of their definite receptors have been reported so far, which seriously restricts the study of its insecticidal mechanism and application. This work identified and confirmed the scavenger receptor class C like protein (Sf-SR-C) acts as the receptor of Vip3Aa protoxin, demonstrated that Sf-SR-C mediates the toxicity of Vip3Aa to Sf9 cells in an internalized manner. These results extend our understanding of SR-C proteins in insects and explain the specificity of Vip3Aa insecticidal activity, which strongly support it as a safe biopesticide. More importantly, it suggests the insecticidal mechanism of Vip3Aa different from the well-known “pore formation” model, “signal transduction” model, as well as newly found “necrosis” model of Cry toxins, which will significantly promote the relevant study of Vip3Aa. Last but not least, because scavenger receptors play a crucial role in innate immunity, our results provide relevant insights into host-pathogen interactions.

Vegetative insecticidal proteins (Vips), which are secreted by some Bacillus thuringiensis strains during vegetative growth, exhibit high virulence to many pests. Vip3A proteins have been used commercially both in some bio-insecticides and in transgenic crops; however, compared with insecticidal crystal proteins, the mechanism of action of Vip3A is still unclear. In this work, we indicated that the fibroblast growth factor receptor-like protein (Sf-FGFR) from the membrane of Sf9 cells could bind to Vip3Aa. The interaction between Vip3Aa and Sf-FGFR was confirmed by pull-down assays and dot blotting experiment in vitro. The binding affinity between Vip3Aa and extracellular regions of Sf-FGFR (GST-FGFR-N) was determined by microscale thermophoresis assay (MST). Moreover, Vip3Aa-Flag could be co-immunoprecipitated with Sf-FGFR-V5 ex vivo. Furthermore, knockdown of Sf-FGFR gene in Sf9 cells resulted in reducing the mortality of those cells to Vip3Aa. In summary, our data indicated that Sf-FGFR is a novel receptor for Vip3Aa.

Vip3Aa, a soluble protein produced by certain Bacillus thuringiensis strains, is capable of inducing apoptosis in Sf9 cells. However, the apoptosis mechanism triggered by Vip3Aa is unclear. In this study, we found that Vip3Aa induces mitochondrial dysfunction, as evidenced by signs of collapse of mitochondrial membrane potential, accumulation of reactive oxygen species, release of cytochrome c, and caspase-9 and -3 activation. Meanwhile, our results indicated that Vip3Aa reduces the ability of lysosomes in Sf9 cells to retain acridine orange. Moreover, pretreatment with Z-Phe-Tyr-CHO (a cathepsin L inhibitor) or pepstatin (a cathepsin D inhibitor) increased Sf9 cell viability, reduced cytochrome c release, and decreased caspase-9 and -3 activity. In conclusion, our findings suggested that Vip3Aa promotes Sf9 cell apoptosis by mitochondrial dysfunction, and lysosomes also play a vital role in the action of Vip3Aa.

Microbially derived, protein-based biopesticides offer a more sustainable pest management alternative to synthetic pesticides. Vegetative insecticidal proteins (Vip3), multidomain proteins secreted by Bacillus thuringiensis, represent a second-generation insecticidal toxin that has been preliminarily used in transgenic crops. However, the molecular mechanism underlying Vip3's toxicity is poorly understood. Here, we determine the distinct functions and contributions of the domains of the Vip3Aa protein to its toxicity against Spodoptera frugiperda larvae. We demonstrate that Vip3Aa domains II and III (DII-DIII) bind the midgut epithelium, while DI is essential for Vip3Aa's stability and toxicity inside the protease-enriched host insect midgut. DI-DIII can be activated by midgut proteases and exhibits cytotoxicity similar to full-length Vip3Aa. In addition, we determine that DV can bind the peritrophic matrix via its glycan-binding activity, which contributes to Vip3Aa insecticidal activity. In summary, this study provides multiple insights into Vip3Aa's mode-of-action which should significantly facilitate the clarification of its insecticidal mechanism and its further rational development.

The vegetative insecticidal proteins (Vips) secreted by Bacillus thuringiensis are regarded as the new generation of insecticidal toxins because they have different insecticidal properties compared with commonly applied insecticidal crystal proteins (Cry toxins). Vip3A toxin, representing the vast majority of Vips, has been used commercially in transgenic crops and bio-insecticides. However, the lack of both structural information on Vip3A and a clear understanding of its insecticidal mechanism at the molecular level limits its further development and broader application. Here we present the first crystal structure of the C-terminal fragment of Vip3A toxin (Vip3Aa11_200–789). Since all members of this insecticidal protein family are highly conserved, the structure of Vip3A provides unique insight into the general domain architecture and protein fold of the Vip3A family of insecticidal toxins. Our structural analysis reveals a four-domain organization, featuring a potential membrane insertion region, a receptor binding domain, and two potential glycan binding domains of Vip3A. In addition, cytotoxicity assays and insect bioassays show that the purified C-terminal fragment of Vip3Aa toxin alone have no insecticidal activity. Taken together, these findings provide insights into the mode of action of the Vip3A family of insecticidal toxins and will boost the development of Vip3A into more efficient bio-insecticides.